Marker for antidepressant therapy and methods related thereto

ABSTRACT

The present invention relates generally to methods for determining the effectiveness of ongoing antidepressant therapy via analysis of the association of G sα  with components of the plasma membrane or cytoskeleton of cells from peripheral tissues of the depressed individual as well as to methods involved in screening for effective antidepressant agents via their ability to cause a difference in the association of G sα  with components of the plasma membrane or cytoskeleton of cells.

[0001] Priority is claimed to U.S. Provisional Appl. No. 60/221,874,filed Jul. 29, 2000, which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to methods for determining theeffectiveness of antidepressant therapy in a depressed individual aswell as methods for detecting agents that possess antidepressantactivity.

[0004] 2. Related Technology

[0005] Affective disorders are characterized by changes in mood as theprimary clinical manifestation. Major depression is one of the mostcommon mental illnesses and is often under diagnosed and frequentlyundertreated, or treated inappropriately. Major depression ischaracterized by feelings of intense sadness and despair, mental slowingand loss of concentration, pessimistic worry, agitation, andself-deprecation. Physical changes usually occur that include insomnia,anorexia and weight loss (or overeating) decreased energy and libido,and disruption of the normal circadian rhythms of activity, bodytemperature, and many endocrine functions. As many as 10-15% ofindividuals with this disorder display suicidal behavior during theirlifetime.

[0006] Antidepressant therapies are present in many diverse forms,including tricyclic compounds, monoamine oxidase inhibitors, selectiveserotonin reuptake inhibitors (SSRIs), atypical antidepressants, andelectroconvulsive treatment. Antidepressant therapies vary widely inefficacy and the response of any given patient to a therapy isunpredictable. Unfortunately, therapy often proceeds for 1-2 monthsbefore it is established whether or not a specific modality of treatmentis effective. Thus, there remains a need for methods of ascertainingwhere the antidepressant therapy is effective in a depressed individualas well as a need for a method of screening for novel antidepressantagents.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to methods for determining theeffectiveness of ongoing antidepressant therapy (during the early stagesof therapy) by whether there has been a modification of the associationof G_(sα) with components of the plasma membrane or cytoskeleton ofcells from peripheral tissues of the depressed individual.

[0008] Another aspect of the invention is directed to methods involvedin screening for effective antidepressant agents via their ability toalter (as compared to a control) the association of G_(sα) withcomponents of the plasma membrane or cytoskeleton of cultured cellsexpressing Type VI adenylyl cyclase.

[0009] Other objectives and advantages of the invention may be apparentto those skilled in the art from a review of the following detaileddescription, including any drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows detergent extraction of G_(sα) from C6-2B gliomamembranes treated with antidepressants.

[0011]FIG. 2 shows co-localization of adenylyl cyclase activity with thepresence of G_(sα) in Triton X-100-extracted C6-2B cells fractionated ona sucrose density gradient.

[0012]FIG. 3 demonstrates that antidepressant treatment of C6-2B cellscauses a shift in the localization of G_(sα) from a TritonX-100-insoluble caveolin-enriched domain to a more Triton X-100-solubledomain.

[0013]FIG. 4 shows fractional distribution of G_(iα) from C6-2B gliomamembranes treated with fluoxetine.

[0014]FIG. 5 demonstrates that chronic desipramine treatment of C6-2Bglioma cells does not alter the overall shape of the cell. Cells weretreated and processed for microscopy as described. A representativeimage of five independent experiments is shown. Bar, 10 μm.

[0015]FIG. 6 shows that chronic desipramine treatment results in anenhancement of G_(sα) immunofluorescence in the cell body and a decreasein the cell processes and process tips. Untreated CD-2B glioma cells (Aand B) display ubiquitous staining of G_(sα) with an enhancement at theprocess tips (arrowheads) and cell processes (asterisks). Desipraminetreated cells C and D) show a decrease in G_(sα) staining at the processtips (arrowheads) and cell processes (asterisks) and simultaneouslydisplay an increase in cell body staining (arrows). Cells were treatedand prepared for microscopy as described previously. Bar=10 μm.

[0016]FIG. 7 shows an enlarged view of the process tips in controlversus desipramine treated C6-2B cells shown in FIG. 6. The process tipsfrom the control cell in FIG. 6A were enlarged to show the intenseG_(sα) staining (A and B) and the corresponding process tips of thedesipramine treated cell in FIG. 6C are shown to demonstrate thereduction of G_(sα) staining after antidepressant treatment © and D).

[0017]FIG. 8 demonstrates the qualitative differences between controland desipramine-treated cells demonstrate a loss of G_(sα) staining inthe cell processes and process tips.

[0018]FIG. 9 shows that G_(oα) does not undergo antidepressant inducedrelocalization. Untreated (A and B) and desipramine-treated © and D)cells show similar G_(oα) immunofluorescence profiles. There is stainingthroughout the cell body and processes of both sets of cells. The figureis typical of approximately 500 cells that were examined. Bar, 10 μm.

[0019]FIG. 10 shows that fluoxetine (10 μM) treatment for 3 days haseffects similar to those of desipramine on G_(sα) cellular localization.Cells were treated with fluoxetine (A) or chlorpromazine (B) andprocessed for confocal microscopy as described. Like desipramine,fluoxetine treatment results in a drastic reduction of G_(sα)immunofluorescence in the cell process (arrow) and process tips(arrowhead), whereas chlorpromazine treatment results in a uniformdistribution of Gsα similar to control (compare to FIGS. 6, A and B).The differential interference contrast image to the right of thefluorescence image shows that these drugs have no effect on global cellshape. Bar, 10 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Despite several decades of studies, the mechanism ofantidepressant action has not been clearly established. One of the mostwidely known biochemical effects of antidepressant treatment is analteration in the density and/or sensitivity of several neurotransmitterreceptor systems [Sulser, Adv. Biochem. Psychopharmacol., 39:249-261;1984]). However, these effects do not fully explain the clinicalefficacy of all antidepressants, mainly because of the dissociationbetween the time course of the change in the receptor numbers and theirclinical time course [Rasenick et al., J. Clin. Psychiatry, 57:49-55;1996].

[0021] Many studies searching for a common mechanism of antidepressantaction have focused on postreceptor neuronal cell signaling processes aspotential targets of such action [Menkes et al. Science, 219:65-76,1983; Ozawa et al., Mol. Pharmacol., 36:803-808, 1989; Duman et al.,Arch. Gen. Psychiatry, 54:597-606, 1997; Takahashi et al., J. Neurosci.,19:610-616, 1999]. Much of this previous work has focused on thedownstream effects of antidepressant action, particularly thoseinvolving cAMP [Perez et al., Eur. J. Pharmacol., 172:305-316, 1989;Perez et al., Neuropsychopharmacology, 4:57-64, 1991; Duman et al.,Arch. Gen. Psychiatry, 54:597-606, 1997; Takahashi et al., J. Neurosci.,19:610-616, 1999]. Our focus is on the upstream events occurring at thepostsynaptic membrane involving G proteins and adenylyl cyclase.

[0022] Much of the current thinking about G protein-coupled receptors isbased on the idea of freely mobile receptors, G proteins, and effectorsin which the specificity of their interaction is derived from thethree-dimensional structure of the sites of protein-proteininteractions. However, recent evidence indicates that an organizedinteraction of receptors, G proteins, and effectors with significantlimitations on lateral mobility [Kuo et al., Science, 260:232-234;1993]. Furthermore, these membrane proteins are associated with tubulinor other cytoskeletal proteins [Carlson et al., Mol. Pharmacol.,30:463-468, 1986; Rasenick et al., Adv. Second Messenger PhosphoproteinRes., 22:381-386, 1990; Wang et al., Biochemistry, 30:10957-10965,1991), which restrict distribution and mobility of G proteins to asurprising degree [Neubig, FASEB, 8:939-946; 1994]. These presence of awell organized network of cytoskeletal elements and the components ofneurotransmitter and hormonal G protein-mediated signal transductionsystems may play an important role in achieving this function.

[0023] Increasing evidence suggests that many species of heterotrimericG proteins are present in caveolin-enriched membrane domains, andcaveolin has been implicated as playing a major role in Gprotein-mediated transmembrane signaling [Okamoto et al., J. Biol.Chem., 273:5419-5422, 1998]. Furthermore, Li et al. [J. Biol. Chem.,270:15693-15701, 1995] reported that the mutational or pharmacologicalactivation of G_(sα) prevents its cofractionation with caveolin. Ourdata indicates that antidepressant treatment of C6-2B cells causes ashift in the localization of G_(sα) from a caveolin-enriched domain to amore Triton X-100-soluble fraction (e.g., see FIG. 3). Such data areconsistent with the our finding that chronic antidepressant treatmentalters the association between G_(sα) and some specific membranecomponent.

[0024] Multiple neural dysfunctions may exist in patients withdepressive disorders, and there are likely to exist multiple moleculartargets for antidepressants. The ability of different classes (datadisclosed herein) of antidepressants to show the same effect on theredistribution of G_(sα) in the plasma membrane indicates convergence.

[0025] In view of the foregoing discussion and by way of illustration ofthe invention, the examples describe methods for determining theeffectiveness of ongoing antidepressant therapy by whether there hasbeen a modification of the association of G_(sα) with components of theplasma membrane or cytoskeleton of cells from peripheral tissues of thedepressed individual as well as methods methods involved in screeningfor effective antidepressant agents via their ability to alter (ascompared to a control) the association of G_(sα) with components of theplasma membrane or cytoskeleton of cultured cells expressing Type VIadenylyl cyclase.

[0026] The invention is illustrated by the following Examples, which arenot intended to limit the scope of the invention as recited in theclaims.

[0027] Example 1 provides methods and materials for experimentsdisclosed in Examples 2-8.

[0028] Example 2 describes results wherein the amount of G_(sα) in C6-2Bglioma cell membranes is not altered by antidepressant treatment.

[0029] Example 3 provides results wherein detergent extraction of G_(sα)from C6-2B glioma membrane is increased by antidepressant treatment.

[0030] Example 4 sets forth results wherein detergent extraction ofG_(sα) from C6-2B glioma membrane is increased by antidepressanttreatment, with disparate antidepressants.

[0031] Example 5 provides results showing that antidepressant treatmentincreases Triton X-100 solubility of G_(sα) from rat synaptic membrane.

[0032] Example 6 provides results with respect to sucrose densitysedimentation of adenylyl cyclase activity in control anddesipramine-treated C6-2B cells

[0033] Example 7 describes results showing that antidepressant treatmentdecreases the colocalization of G_(sα) with triton-insoluble,caveolin-enriched membrane domains and antidepressant-enhanced mobilityis unique to G_(s).

[0034] Example 8 sets forth results of experiments showing thatantidepressant-enhanced mobility is unique to G_(sα).

[0035] Example 9 provides methods and materials for experimentsdisclosed in Examples 10-13.

[0036] Example 10 sets forth results from experiments showing thatchronic antidepressant leads to a shift in cellular localization ofG_(sα).

[0037] Example 11 provides results from experiments that show thatantidepressant-induced G protein a subunit cellular relocalization isspecific to G_(sα).

[0038] Example 12 sets forth results that show that (1) fluoxetinetreatment also promotes G_(sα) migration and (2) chlorpromazine did notinduce migration. Example 13 sets forth methods for screening for theeffectiveness of antidepressant therapy as well as screening for agentshaving antidepressant activity.

EXAMPLE 1 METHODS AND MATERIALS

[0039] Set forth below are methods and materials for experimentsdisclosed in Examples 2-8.

[0040] CELL/TISSUE PREPARATION

[0041] C6-2B cells (between passages 20 and 40) were grown in 175-cm²flasks in Dulbecco's modified Eagle medium, 4.5 g of glucose/L, 10%bovine serum, in a 10% CO₂ atmosphere, at 37° C. for 3 days aftersplitting. As 5 μM for 5 days and 10 μM for 3 days of antidepressanttreatment had a similar effect on the Gpp(NH)p- or forskolin-stimulatedadenylyl cyclase activity in the C6-2B cells [Chen et al., J.Neurochem., 64:724-732, 1995], the latter paradigm was used and cellswere treated with 10 μM drug for 3 days. Media containing drugs(fluoxetine, amtriptyline, iprindole, desipramine, or chlorpromazine)were added to different flasks, and the media were changed daily. Duringthe period of exposure to antidepressants, no morphological change inthe cells was observed. After treatment, the cells were incubated indrug-free media for 1 h before harvesting by scraping with a rubberpoliceman in HEPES-sucrose buffer [15 mM HEPES, 0.25 M sucrose, 0.3 mMphenylmethylsulfonyl fluoride (PMSF), 1 mM EGTA, and 1 mM dithiothreitol(DTT), pH 7.5], C6-2B membranes were prepared as described [Rasenick andKaplan, FEBS Lett. 207:296-301, 1986] and stored under liquid N₂ untiluse. Male Sprague-Dawley rats weighing 150-200 g were fed ad libitum andmaintained in a 12-h light/dark cycle. The method of antidepressanttreatment has been described previosuly [Ozawa and Rasenick, Mol. Pharm.36:803-838, 1989]. In brief, animals were treated with desipramine orfluoxetine (10 mg/kg i.p.) once daily for 21 days; the control groupreceived only saline injection daily for 21 days. Rat cerebral cortexmembranes were prepared according to the method of Rasenick et al.[Nature, 294:560-562; 1981].

[0042] MEMBRANE PROTEIN EXTRACTION

[0043] Membrane proteins were extracted sequentially from C6-2B or ratsynaptic membranes as described [Yan et al., J. Neurochem.,66:1489-1495; 1996]. These membranes were stirred on ice in HEPES (15mM, pH 7.4) containing 1% Triton X-100 for 60 min followed bycentrifugation at 100,000 g for 60 min at 4° C. The supernatant wasreserved (Triton X-100 extract), and the resulting pellet wasresuspended in Tris (15 mM, pH 7.4) containing 1.4% Triton X-114 and 150mM NaCl. The solution was stirred for 60 min at 4° C. and centrifugedfor 30 min at 100,000 g in the cold. The supernatant was saved (TritonX-114 extract). These supernatants and remaining pellet (remainder) weresubjected to sodium dodecyl sulfate (SDS)-polyacrylamide gelelectophoresis (PAGE) using 10% gels. All of the extraction bufferscontained 1 mM PMSF and 1 mM EGTA.

[0044] CELL FRACTIONATION BY SUCROSE DENSITY GRADIENT SEDIMENTATION

[0045] C6-2B cells treated as described above were used to prepareTriton-insoluble, caveolin-enriched membrane fractions by the procedureof Li et al. [J. Biol. Chem., 270:15693-15701; 1995] with minormodifications. In brief, C6-2B cells were harvested into 0.75 ml ofHEPES buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.3 mM PMSF)containing 1% Triton X-100. Homogenization was carried out with 10strokes of a Potter-Elvehjem homogenizer. The homogenate was adjusted to40% sucrose by the addition of an equal volume of 80% sucrose preparedin HEPES buffer and placed at the bottom of an ultracentrifuge tube. Astep gradient containing 30, 15, and 5% sucrose was formed above thehomogenate and centrifuged at 50,000 rpm for 20 h in an SW65 rotor(240,000 g). Two or three opaque bands confined between the 15 and 30%sucrose layers were harvested, diluted threefold with HEPES buffer, andpelleted in a microcentrifuge at 16,000 g. The pellet was resuspended inHEPES buffer and identified as the Triton-insoluble fraction. The 40%sucrose region of the gradient was saved as the Triton-soluble fraction.In separate experiments, the same conditions were used, but instead ofisolating the three opaque bands, 100-μl fractions were collected andassayed for G, content and adenylyl cyclase activity.

[0046] ASSAY OF ADENYLYL CYCLASE ACTIVITY

[0047] Adenylyl cyclase was assayed as described previously [Rasenick etal., Brain Res., 488:105-113; 1989]. Each sucrose gradient fraction wasassayed under both basal and stimulated (10⁻⁴M forskolin) conditions for10 min at 30° C. in 100 μl of medium containing 15 mM HEPES, pH 7.5,0.05 mM ATP, [α-³²P]ATP (5×10⁶ cpm/tube), 5 mM MgCl₂, 1 mM EGTA, 1 mMDTT, 0.5 mM cyclic AMP (cAMP), 60 mM NaCl, 0.25 mg/ml bovine serumalbumin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 U of adenosinedeaminase/ml, and a nucleotide triphosphate regenerating systemconsisting of 0.5 mg of creatine phosphate, 0.14 mg of creatinephosphokinase, and 15 U of myosin kinase/ml. The reaction was stopped byaddition of 0.1 ml of a solution containing 2% SDS, 1.4 mM cAMP, and 40mM ATP, and the [³²P]cAMP formed was isolated by the method of Salomon(1979) using [³H]cAMP to monitor recovery. All assays were performed intriplicate.

[0048] IMMUNOBLOTTING

[0049] Rat cortex membrane, C6-2B cell membranes, or Triton extracts ofeach membrane preparation were subjected to SDS-PAGE followed byelectrotransfer to polyvinylidene difluoride (PVDF) membrane. The PVDFmembrane was incubated with a phosphate-buffered saline/Tween 20 Buffer(140 mM NaCl, 27 mM KCl, 81 mM Na₂HPO₄, 15 mM KH₂PO₄, 0.1% Tween 20, pH7.4) and 3% bovine serum albumin. The membrane was then incubated withpolyclonal rabbit antisera against the various G protein subunits [RM(G_(sα)) or 116 (G_(i1), G,_(i2), G,_(i3α))] at a 1:25,000 (RM) or1:5,000 (116) dilution (see below for source of antibodies). The PVDFmembranes were washed three times and incubated with a dilution of1:5,000 of the second antibody [horseradish peroxidase-linkedanti-rabbit IgG F(ab′)₂ (Amersham)]. Immunoreactivity was detected withan enhanced chemiluminescence (ECL) western blot detection system(Amersham) in accord with the manufacturer's instructions. The developedautoradiographs were analyzed by densitometry.

[0050] G PROTEIN PURIFICATION AND QUANTIFICATION

[0051] G_(sα) (His) His₆ was purified, from Escherichia coli expressingthe recombinant gene for that protein, by a modification of the methodof Gilman [Lee et al., Methods Enzymol., 237:146-164; 1994]. In brief,the bacteria were grown overnight in an enriched medium (2% tryptone, 1%yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mM KH₂PO₄, pH 7.2)containing 50 μg/ml ampicillin 8 L (8×1 L in 2-L Erlenmeyer flasks).Bacteria were collected by centrifugation, cells were lysed bysonication, the cleared cell lysate was loaded onto a Ni-NTA resincolumn (QiaGen), and the protein was eluted with a step gradient of 20to 60 mM imidazole. G_(sα) protein was purified further byhigh-performance liquid chromatography with Resource Q chromatography(Pharmacia) and hydroxylapatite chromatography. Fractions containingG_(sα) protein were identified by labeling with[α-³²P]P³-(4-azidoanalido)-P¹-5′-GTP and immunoblotting. The G_(sα)was >98% pure as identified by silver stain of the gels.

[0052] Purified recombinant G_(sαL) (long form of G_(sα); 1-10 ng/lane)was subjected to SDS-PAGE and transferred to PVDF membranes. The blotswere incubated with antibody against G_(sα) (RM) and processed by ECL.Films were analyzed on a Molecular Dynamics densitometer, and the volumeof each band was quantified and used to make a standard curve. psMATERIALS AND DATA ANALYSIS

[0053] All detergents were obtained from Pierce, Anti-G proteinantibodies were from Drs. David Manning (116; University ofPennsylvania) and Allen M. Spiegel (RM; National Institutes of Health).Caveolin antibody was obtained from Transduction Laboratories(Lexington, Ky., U.S.A.) clone no 2297. All other reagents used were ofanalytical grade.

[0054] Further, antibodies for use in the protocols disclosed herein mayalso be manufactured by well known methods [Antibodies, A LaboratoryManual, Harlow and Lane, Cold Spring Harbor Laboratory; ISBN:0879693142].

[0055] Data were analyzed for statistical significance using Scheffe'stest or Bonferroni's multiple comparison test after a one-way ANOVA testor two-tailed t test. Values of p<0.05 were taken to indicatesignificance.

EXAMPLE 2 THE AMOUNT OF G_(Sα) IN C6-2B GLIOMA MEMBRANE IS NOT ALTEREDBY ANTIDEPRESSANT TREATMENT

[0056] Experiments were conducted to determine whether G_(sα) isquantitatively altered by antidepressant therapy. For quantification ofG_(sα) in C6-2B membrane, a standard curve was created using purifiedrecombinant G_(sα). Pure G_(sα) (1-20 ng/well) was subjected to SDS-PAGEand transferred to a PVDF membrane, G_(sα) was immunodetected byantisera against the as subunit of G protein.

[0057] To estimate the effect of antidepressant treatment on the amountof G_(sα) in C6-2B membranes, equal amounts (10 μg) of C6-2B gliomamembrane proteins in control and chronically (3 days)antidepressant-treated groups were subjected to SDS-PAGE. G_(sα) wasimmunodetected by antisera against the as subunit of G protein. Table 1summarizes the quantity of G proteins in the membrane of C6-2B cellsafter exposure to 10 μM (3 days) amitriptyline, iprindole, or fluoxetine(values are expressed as means ±standard error of the mean of 4-5experiments. As shown in Table 1, no significant differences weredetected (p>0.05) between antidepressant-treated and control groups.TABLE 1 Effect of antidepressant treatment on the content of G proteinin C6-2B glioma membranes Subunit of G protein amount (ng/10 μg ofmembrane protein) G protein Control Iprindole Amitriptyline FluoxetineαsL  8.35 ± 0.05  8.20 ± 0.20  8.13 ± 0.17  7.23 ± 0.56 αsS  5.15 ± 0.28 4.70 ± 0.12  4.98 ± 0.21  5.65 ± 0.74 Total 13.50 ± 0.27 12.93 ± 0.3613.10 ± 0.34 12.49 ± 0.67

EXAMPLE 3 DETERGENT EXTRACTION OF G_(Sα) FROM C6-2B GLIOMA MEMBRANE ISINCREASED BY ANTIDEPRESSANT TREATMENT

[0058] In order to determine whether detergent extraction of G_(sα) fromcells treated with antidepressants increased the following experimentwas undertaken.

[0059] C6-2B cells were treated chronically with iprindole,amitriptyline, or chlorpromazine (10 μM, 3 days) and harvested. Amembrane-enriched fraction was prepared (see Example 1), and membraneproteins were extracted sequentially with Triton X-100 (Tx 100) andTriton X-114 (Tx 114). Equal amounts of these extracts were subjected toSDS-PAGE and transferred to PVDF membrane. The long and short forms ofG_(sα) (G_(sαL) and G_(sαS)) from different fractions were identified byimmunodetection. A representative immunoblot (FIG. 1) shows theredistribution of G_(sαL) and G_(sαS) in the plasma membrane afterchronic antidepressant treatment (i.e., the effect of the tricyclicantidepressant (amitriptyline) and an atypical, non-reuptake-inhibitingantidepressant (iprindole) on the redistribution). The data indicateG_(sα) was shifted to the less hydrophobic fraction (Triton X-100extract) from the more hydrophobic fraction (Triton X-114 extract)subsequent to treatment with antidepressant G_(sα) exists in four splicevariations that migrate as a long form (G_(sαL)) and short form(G_(sαS)) (the two long and two short variants are not resolved from oneanother). G_(sαL) in the Triton X-114 fraction was significantly lowerin the iprindole- and amitriptyline-treated groups than in the controlgroup. G_(sαS) also showed a tendency to migrate to the less hydrophobicdomain (Triton X-100 extract) from a more hydrophobic domain of theplasma membrane.

EXAMPLE 4 DISPARATE ANTIDEPRESSANTS HAVE SIMILAR EFFECTS

[0060] Experiments were conducted according to the method set forth inExample 1 to assess the effects of different antidepressants on thedetergent extraction of G_(sα) from C6-2B glioma cells. Three differentantidepressants comprised of the tricyclic antidepressant(amitriptyline), the non-reuptake inhibitor (iprindole), and SSRI(fluoxetine) were used. Membranes were prepared from C6-2B glioma cellsthat had been exposed to a 10 μM concentration of the indicatedantidepressant drug for 3 days.

[0061] The ratios of the percentage of G_(sα) extracted by the twodetergents in the treatment versus control groups were compared and areset forth in Table 2 as percentage of control (i.e., each Triton X-100extract was compared with the untreated Triton X-100 extract; the samewas done for Triton X-114 extracts as well as the remainder). The valuesshown are means ±standard error of the mean of 4-5 experiments.

[0062] The different antidepressants achieved similar effects on G_(sα),i.e., G_(sα) shifted to the less hydrophobic, Triton X-100 fractionsubsequent to antidepressant treatment. In contrast, chlorpromazine,which is a tricyclic compound but not an antidepressant, did not exertthese effects. The total amount of G_(sα) was not changed byantidepressant treatment, membrane preparation, or detergent extraction.

[0063] Other experiments showed that amphetamine, which is known toblock neurotransmitter uptake but does not have antidepressant activity,was also without effect. TABLE 2 Effects of antidepressants on detergentextraction of G_(sα) from C6-2B cells % of corresponding value incontrol group Extraction G_(sα) Antidepressant subunit Triton X-100Triton X-114 Remainder Iprindole G_(sαL) 128.0 ± 3.8^(a)  75.3 ± 2.2^(b) 87.7 ± 9.3 G_(sαS) 146.6 ± 13.3^(b)  74.7 ± 0.9^(b) 112.0 ± 1.9^(a)Amitriptyline G_(sαL) 120.3 ± 3.7^(b)  84.5 ± 0.8^(b)  83.4 ± 14.5G_(sαS) 140.3 ± 9.3^(b)  92.7 ± 3.8  82.6 ± 5.1^(a) Fluoxetine G_(sαL)146.4 ± 12.7^(a)  84.4 ± 2.9^(b)  90.8 ± 34.2 G_(sαS) 215.9 ± 30.9^(a) 92.2 ± 1.3^(b)  99.7 ± 3.1 Chlorpromazine G_(sαL) 107.4 ± 8.3 105.7 ±6.5  83.8 ± 22.0 G_(sαS) 113.4 ± 10.7 102.2 ± 15.6 100.4 ± 31.6

EXAMPLE 5 ANTIDEPRESSANT TREATMENT INCREASES TRITON X-100 SOLUBILITY OFG_(Sα) FROM RAT SYNAPTIC MEMBRANE

[0064] Rats were treated with antidepressant via intraperitonealinjection once daily for 21 days, control animals received an equalnumber and volume of injection of saline. 20 Cerebral cortices from eachgroup were removed. Rat synaptic membrane-enriched fractions wereprepared (see Example 1). The ratios of the percentage of G_(sα)extracted by the two detergents in the treatment versus control groupswere compared and are set forth in Table 3 as percentage of control (i.e., each Triton X-100 extract was compared with the untreated TritonX-100 extract, the same was comparison was undertaken for Triton X-114extracts as well as the remainder). The values shown are means ±standard error of the mean of 4-6 experiments.

[0065] Results indicate that both desipramine and fluoxetine treatmentcaused a significant increase of G_(sαL) in the Triton X- 100 extractand a concomitant decrease in that protein in the Triton X-114 extract.Under no circumstances did the antidepressant treatment alter the amountof G_(sα). The sum of G_(sα) inmnunoreactivity of the three fractionswas not changed. Previous studies had demonstrated that a 1-daytreatment of cells or a 1-week treatment of rats was without effect inany of the parameters examined [Ozawa et al., J. Neurochem., 56:330-338,1991; Chen et al., J. Neurochem., 64:724-732, 1995]). Similar short-termtreatments with fluoxetine were also without effect. TABLE 3 Effects ofchronic antidepressants on detergent extraction of G_(sα) from ratsyanptic membrane % of corresponding value in control group ExtractionAntidepressant G_(sα) treatment Subunit Triton X-100 Triton X-114Remainder Desipramine G_(sαL) 128.2 ± 9.5^(a) 77.3 ± 8.8^(a) 66.0 ±10.2^(a) G_(sαS) 146.4 ± 9.6^(b) 71.7 ± 6.8^(a) 84.9 ± 25.7 FluoxetineG_(sαL) 118.1 ± 3.4^(a) 81.9 ± 4.5^(a) 53.8 ± 6.9^(a) G_(sαS) 153.8 ±39.9 91.6 ± 6.3 45.2 ± 8.4^(a)

EXAMPLE 6 SUCROSE DENSITY SEDIMENTATION OF ADENYLYL CYCLASE ACTIVITY INCONTROL AND DESIPRAMINE-TREATED C6-2B CELLS

[0066] Membranes from control and 10 μM desipramine-treated C6-2B gliomacells were solubilized in Triton X-100 and run on a discontinuoussucrose density gradient. Fractions were collected and assayed foradenylyl cyclase activity and for the presence of G_(sα) by SDS-PAGE andimmunoblotting.

[0067]FIG. 2A shows adenylyl cyclase activity was measured on sucrosedensity gradient fractions from control (circles) anddesipramine-treated (squares) C6-2B cells under basal (open symbols) andforskolin-stimulated (filled symbols) conditions. FIG. 2B shows a G_(sα)imrnunoblot corresponding to the assayed fractions in FIG. 2A.

[0068]FIG. 2A demonstrates an increase in forskolin-stimulated adenylylcyclase activity for both control and desipramine-treated cells (notethe corresponding increase in G_(sα) and adenylyl cyclase activity infractions 10 and 12 of the desipramine-treated group). Further, there isalmost a twofold increase in enzyme activity in the desipramine-treatedcells compared with control in fractions 11 and 12. Basal adenylylcyclase activity is unchanged in either group. The increase inforskolin-stimulated adenylyl cyclase activity corresponds to anincrease in G_(sα) in these same fractions (FIG. 2B, fraction 12).

EXAMPLE 7 ANTIDEPRESSANT TREATMENT DECREASES THE COLOCALIZATION OFG_(Sα) WITH TRITON-INSOLUBLE, CAVEOLIN-ENRICHED MEMBRANE DOMAINS ANDANTIDEPRESSANT-ENHANCED MOBILITY IS UNIQUE TO G_(S)

[0069] Recently, it has been reported that caveolin, a Triton-insolublemembrane protein, participates in plasma membrane coupling events(Okamoto et al., J. Biol. Chem., 273:5419-5422; 1998). To evaluate theinteraction between G_(sα) and caveolin-enriched membrane domains, C6-2Bcell lysates were subjected to sucrose density gradient fractionationand divided into a low-density, Triton X-100-insoluble fraction, whichis dramatically enriched in caveolin, and a Triton X-100-solublefraction, which contains the majority of other membrane proteins.Results are set forth in FIG. 3.

[0070]FIG. 3A reveals a set of representative immunoblots of thedistribution of G_(sα) after sucrose density gradient fractionation. Asshown, chronic antidepressant treatment of C6-2B cells causes a shift inthe localization of G_(sα)from a caveolin-enriched domain to a moreTriton X-100-soluble domain. The amount of G_(sα) in the solublefraction of desipramine- and fluoxetine-treated cells was consistentlythree to five times greater than the amount in the caveolin-enrichedfraction, making a direct measurement of the shift from one domain tothe other nearly impossible. FIG. 3C shows the percent change in G_(sα)in the caveolin-enriched Triton-insoluble fraction normalized to equalcaveolin. FIG. 3B shows a typical caveolin immunoblot used for thisnormalization. There was only a 5-10% difference in the amount ofcaveolin isolated in the Triton-insoluble domain between theexperimental groups, and very little caveolin was found in the solublefraction. Both desipramine and fluoxetine treatment decreased the amountof G_(sα) in the caveolin-enriched fraction by ˜50%. In contrast,chlorpromazine caused a reduction of no more than 25% of G_(sα) in thecaveolin-enriched fraction. Results were determined to be significant byone-way ANOVA (***p<0.0001), as well as Bonferroni's (***p<0.001)multiple comparison tests.

EXAMPLE 8 ANTIDEPRESSANT-ENHANCED MOBILITY IS UNIQUE OF G_(sα)

[0071] Experiments were conducted (as described in Example 1) toascertain the effect of antidepressant therapy on the mobility of G_(iα)from C6-2B glioma cells. Specifically, C6-2B cells were treated withfluoxetine (10 μM, 3 days) and harvested. A membrane-enriched fractionwas prepared, and membrane protein was extracted sequentially withTriton X-100 (Tx 100) and Triton X-114 (Tx 114). Equal amounts of theseextracts were subjected to SDS-PAGE and transferred to PVDF membrane.The PVDF membrane was incubated with polyclonal rabbit antisera againstG_(iα1), and G_(iα2a). Immunoreactivity was detected with an ECL westernblot detection system.

[0072] Results, which are set forth in FIG. 4, indicated that unlikeG_(sα), G_(iα) did not migrate to a less hydrophobic membrane fractionafter antidepressant treatment.

EXAMPLE 10 METHODS AND MATERIALS

[0073] Set forth below are method and materials used in experimentsdiscussed in Examples 10-13.

[0074] CELL CULTURE

[0075] C6-2B cells (between passages 30 and 50) were plated ontocoverslips and allowed to attach overnight in Dulbecco's modifiedEagle's medium, 4.5 g/l glucose, 10% bovine serum, and 100 μg/mlpenicillin and streptomycin at 37° C. in a humidified 10% CO₂atmosphere. As reported previously, desipramine treatment regimens of 3μM for 5 days and 10 μM for 3 days yielded similar biochemical results(Chen and Rasenick, 1995b). Therefore, the latter treatment paradigm wasused in these experiments because it was easier to maintain the cellcultures for 3 days. In some instances, 10 μM fluoxetine was used. Theculture media and drug were changed daily. Neither desipramine norfluoxetine treatment altered cell growth (as determined by theconfluence of the cell monolayer and total protein estimation) or cellviability (as determined by 4,6-diamidino-2-phenylindole staining andvisualization under a fluorescence microscope with UV light). During thetreatment duration, no morphological changes were observed in the cells.After the treatment duration, the cells were incubated in drug-freemedia for 45 to 60 minutes before fixation.

[0076] INDIRECT IMMUNOFLUORESCENCE LASER SCANNING CONFOCAL MICROSCOPY

[0077] After treatment, cells were washed once with phosphate-bufferedsaline (PBS, 136 nM NaCl, 2.6 mM KC1, 5.4 mM Na_(a)PO₄7H₂O, pH 7.4) andfixed with ice-cold methanol for 10 minutes. Cells were then washedthree times with PBS followed by 2 h of blocking in 5% normal goatserum/0.2% fish skin gelatin in PBS. Primary antibody was added for 1.5h, G_(sα)/RM1 (PerkinElmer Life Sciences, Boston, Mass.) 1:50 and Goα(Santa Cruz Biotechnology, Santa Cruz, Calif.) 2 μg/ml, followed bythree washes with PBS. Oregon Green-labeled secondary antibody(Molecular Probes, Eugene, Oreg.) was added at a concentration of 8μg/ml for 1 h followed by three PBS washes. The coverslips were mountedonto slides with Vectashield (Vector Laboratories, Burlingame, Calif.)containing diamidino-2-phenylindole as a mounting medium. Images wereacquired using a Zeiss LSM510 laser-scanning confocal microscope (CarlZeiss Inc., Thornwood, N.Y.). A single 488-nm beam from an argon/kryptonlaser was used for excitation of the Oregon Green. Differentialinterference contrast images were also acquired. Five experiments wereperformed and coverslips were examined. Approximately 2100 cells fromcontrol and desipramine-treated coverslips were counted by twoinvestigators blind to the experimental conditions over the course ofthe five experiments.

[0078] FLUORESCENCE QUANTIFICATION

[0079] The cellular distribution of G_(sα) was quantified in confocalimaged C6-2B cells using NIH-Image software(http://rsbinfonihgov/nih-image) as describe previously [Southwell etal., Cell Tissue Res., 292:37-45, 1998; Jenkinson et al., Br. J.Pharmacol., 126:131-136, 1999]. Images of 9×1 μm optical, planarsections taken from four randomly selected control and four randomlyselected desipramine-treated cells were captured and the middle fivesections from each cell were quantified. Total cellular G_(sα)fluorescence was measured by counting the number of pixels withintensity above threshold (determined by minimum intensity abovebackground in this case 50 pixels). The areas of intensity were numberedand divided visually into those localized to the cell body and thoselocalized to the processes and process tips. The total from each regionwas divided by the total cell pixel intensity and expressed as apercentage of the total. This was done for each section of each cell andthe sections were averaged per cell to give an average percentage totalper cell.

[0080] In a separate investigation, seven sets of 300 cells each fromcontrol group and desipramine-treated cells from five experiments werecounted to determine the primary localization (processes and processtips or cell body) of G_(s)α within these cells. The majority of thecells stained positively for G_(sα) throughout the entire cell, butthere was usually an enhancement in one of these regions. Overlyflattened and fragmented cells were omitted from counting, as were cellsthat did not display processes. The counts are expressed as the ratio ofprocess and process tip localization/cell body localization.

[0081] DATA ANALYSIS

[0082] Images were evaluated by two investigators blinded to thetreatment condition. Student's t test was performed for statisticalanalysis. Values of p<0.05 were taken to indicate significance.

EXAMPLE 10 CHRONIC ANTIDEPRESSANT TREATMENT LEADS TO A SHIFT IN THECELLULAR LOCALIZATION OF G_(sα)

[0083] Studies were conducted to determine whether a shift in cellularlocalization of G_(sα) occurred in C6-2B cells subjected toantidepressants. C6-2B glioma cells were treated with the tricyclicantidepressant desipramine (10 μM) for three days and were then examinedby laser scanning confocal microscopy to visualize these changes inmembrane localization (See Example 9 for specific experimentalprotocol).

[0084] Examination of 300 to 500 control and desipramine-treated cellsby three independent researchers revealed that desipramine treatment didnot alter the overall structure of C6-2B cells (FIG. 5), but drasticallyreduced the presence of G_(sα) in the process tips (FIG. 6, arrowheadsand FIG. 7). In addition, there was an increase in the presence ofG_(sα) within the cell body of many of the desipramine-treated cells(FIG. 6, arrows), as well as a decrease within the cell processesthemselves (FIG. 6, asterisks). In some instances, there was an intenseclustering of G_(sα) staining in the cell body (FIG. 6C, arrows), butthe majority of the cells did not exhibit such a focal increase inG_(sα) staining.

[0085] Twenty-one hundred cells from each group (control versusdesipramine-treated) over a series of five experiments were examined toquantify the extent of the antidepressant effect. The cells were groupedinto two categories: those that displayed intense staining at theprocess tips as well as overall staining in the processes and cell body(category A) versus those that displayed intense staining in the cellbody region and decreased process and process tip staining (category B).Abnormal cells or those not displaying processes were not included inthe cell count. Cells (300-450) were counted per experiment and theratio of category A cells to category B cells for each group is shown inFIG. 8.

[0086] Twice as many control cells (64%) displayed G_(sα) staining atthe process tips and throughout the entire cell than those treated withdesipramine (32%). This demonstrates that G_(sα) relocalization is notan all-or-none response to antidepressant treatment and that some cellsmay be more responsive to treatment than others.

[0087] To determine quantitative differences between the groups, five 1μm optical, planar sections through each of four cells in each groupwere examined by confocal microscopy and the digital images werecaptured. These images were then analyzed using the program NIH Image[Southwell et al., Cell Tissue Res., 292:37-45, 1998; Jenkinson et al.,Br. J. Pharmacol., 126:131-136, 1999]. These experiments were undertakento account for changes in G_(sα) localization at different focal planesof the cell. The percentage of G_(sα) localized to the cellularprocesses and process tips of control versus treated cells were comparedby dividing the pixel density above threshold in these regions by thetotal cellular pixel density (Table 4). There was a three-fold decreasein G_(sα) localization to the processes and process tips between controlcells and desipramine-treated cells as 12% of the total cellular G_(sα)was located in the process tips of control cells versus 4% present inthe tips after desipramine treatment. TABLE 4 Percentage of G_(sα)Immunofluorescence Distribution in the Cell Sample Cell Body ProcessesControl C1 83.9 16.1  C2 89.0 11.0  C3 83.5 16.5  C4 95.6 4.4 Average88.0 ± 5.7 12.0 ± 5.7 Desipramine D1 91.4 8.6 D2 92.2 7.8 D3 100.0  0.0D4 100.0  0.0 Average 95.9 ± 4.7  4.1 ± 4.7

EXAMPLE 11 ANTIDEPRESSANT INDUCED G PROTEIN α SUBUNIT CELLULARRELOCALIZATION IS SPECIFIC TO G_(sα)

[0088] To determine whether antidepressant-induced mobility is specificto G_(sα), G_(oα) distribution was examined in approximately 500 cellsunder the same treatment conditions. FIG. 9 shows that there was littleif any change in the distribution of G_(oα) after desipramine treatment.G_(oα) is throughout the cell without specific regions displaying anincreased staining intensity in control or treated cells. Some of thecontrol cells (FIGS. 9A and 9B) have a slight increase in stainingintensity at the process tips, but this is also seen in the treatedcells (FIGS. 9C and 9D), indicating that antidepressant treatment doesnot effect G_(oα) localization within the cell.

EXAMPLE 12 FLUOXETINE TREATMENT ALSO PROMOTES G_(sα) MIGRATION

[0089] If the redistribution of G_(sα) is truly an antidepressanteffect, then other classes of antidepressant drug should have a similareffect. Confocal microscopic images of C6-2B cells treated with 10 μMfluoxetine for three days show a similar Gsμ staining pattern comparedwith desipramine-treated cells (FIG. 6A). The most striking similarityof desipramine and fluoxetine effects on G_(sα) localization is the lossof staining in the processes and process tips (compare FIGS. 6C and 6D,and FIG. 10A with FIGS. 6A and 6B). Approximately 100 cells wereexamined for qualitative differences as described above for FIG. 8. Ofthe fluoxetine-treated cells, 45% displayed intense staining in theprocess tips compared with the 64% of control and 32% ofdesipramine-treated cells mentioned previously.

[0090] CHLORPROMAZINE

[0091] The antipsychotic drug chlorpromazine was used as a control forantidepressant effects. When cells were treated with 10 μMchlorpromazine for three days, G_(sα) staining was evident throughoutthe cell body (FIG. 10B): there is G_(sα) immunostaining throughout thecell body, cell process, and process tip. This pattern of G_(sα)distribution was similar to other control cells; 68% of approximately100 cells demonstrated distinct staining in the cell processes andprocess tips.

[0092] OTHER TREATMENT PARADIGMS HAVE A SIMILAR EFFECT ON G_(sα)

[0093] A lower dosage and longer exposure time for desipramine treatment(3 μM for five day) was also tested. Control cells have intense stainingat the process tips, whereas the desipramine treated cells do not. Themain difference between the high-dose/three-day and thelow-dose/five-day treatment regimens is the cell body localization ofG_(sα). A majority of C6-2B cells treated with 10 μM desipramine displayintense clustering of GSA in the perinuclear region whereas cellstreated with 3 μM desipramine show a more even distribution betweenintense cell body staining and a more nondescript staining. One-day/10/μM desipramine treatment of C6-2B cells resulted in a G_(sα)distribution similar to cells treated with 3 μM for five days (data notshown). Under the acute treatment condition (one day, 10 μM) the numberof cells lacking G_(sα) in the process tips was not significantlydifferent from the control cell population seen in Table 4 and FIG. 8

EXAMPLE 13 SCREENING FOR EFFECTIVENESS OF ANTIDEPRESSANT THERAPY ANDSCREENING FOR AGENTS HAVING ANTIDEPRESSANT ACTIVITY

[0094] In view of the foregoing results with respect to modificationsnoted in the association of G_(sα) with components of the plasmamembrane or cytoskeleton from glioma cells, this example is directed toa method for detecting the effectiveness of antidepressant therapy aswell methods for screening for agents having antidepressant activity.

[0095] An individual, diagnosed with major depression and receivingantidepressant therapy, may be assessed for the effectiveness of suchtherapy by the following method. Cells, for example, but not limited toblood cells [erythrocytes (red cells), leukocytes (white cells),platelets] and skin fibroblasts from peripheral tissues of the depressedindividual are collected and a determination is made as to whether therehas been a modification of the association of G_(sα) with components ofthe plasma membrane or cytoskeleton of cells from peripheral tissues ofthe depressed individual. Such modifications may include, but are notlimited to enhanced coupling between G_(sα) and adenylyl cyclase,redistribution of G_(sα) from a strongly hydrophobic region of theplasma membrane to a less hydrophobic membrane domain, and/orredistribution of G_(sα) from cell processes and process tips to thecell body.

[0096] Antidepressent therapy often requires about one month to begin toachieve effectiveness. Often multiple drugs must be employed before asatisfactory combination is stumbled upon. Any difference in suchmodifications (as compared to a normal or control state), when noted inthe early stages of antidepressant therapy and correlated with asubsequent decrease in the clinical depressive state would serve toquickly predict the success and/or failure of antidepressant therapy.More specifically, unlike psychological tests, if antidepressant therapywere to be effective, it is likely to increase such modifications in theassociation of G_(s)α with components of the plasma membrane orcytoskeleton within 3-5 days.

[0097] Further, the present invention is also useful for determining theeffectiveness of a putative antidepressant agent or agents, in that suchcompounds may be rapidly screened using the methods described herein.Specifically, putative agents are introduced in a cell culture whereinthe cells (for example, but not limited to Neuro2A cells(neuroblastoma), SKNSH cells (human blastoma) HEK293 cells (humanembroynic kidney cells after transfection with Type VI adenylyl cyclase)express Type VI adenylyl cyclase and a determination is made as towhether there has been a modification (for example, but not limited toenhanced coupling between G_(sα) and adenylyl cyclase, redistribution ofG_(sα) from a strongly hydrophobic region of the plasma membrane to aless hydrophobic membrane domain, and redistribution of G_(sα) from cellprocesses and process tips to the cell body of the association of G_(sα)with components of the plasma membrane or cytoskeleton of cells. Thoseagents that would be expected to have antidepressant activity would bethose compounds that increase the modifications in the association ofG_(sα) with components of the plasma membrane or cytoskeleton.

[0098] Further, the foregoing process lends itself to the use offluorescence resonance energy transfer (FRET) techniques for use as ahigh throughput systems. We have recently developed a fluorescent analogof G_(sα) (a GPP fusion protein) for use in our experiments. Such ananalog is used with a fluorescent adenylyl cyclase to determine theeffects an antidepressant on the modification of interaction between Gsαand adenylyl cyclase. Specifically and in view of the foregoing examplesand discussion, if the agent in question possessed antidepressantactivity one would see an increase in FRET (an increased interactionbetween the fluorescent analog of G_(sα) and fluorescent adenylylcyclase).

[0099] Although the present invention has been described in terms ofpreferred embodiments, it is intended that the present inventionencompass all modifications and variations that occur to those skilledin the art upon consideration of the disclosure herein, an in particularthose embodiments that are within the broadest proper interpretation ofthe claims and their requirements.

[0100] All literature cited herein is incorporated by reference.

We claim:
 1. A method for detecting the effectiveness of antidepressanttherapy in a depressed individual comprising determining whether therehas been a modification of the association of G_(sα) with components ofthe plasma membrane or cytoskeleton of cells from peripheral tissues ofthe depressed individual.
 2. The method of claim 1 wherein themodification is enhanced coupling between G_(sα) and adenylyl cyclase.3. The method of claim 1 wherein the modification is a redistribution ofG_(sα) from a strongly hydrophobic region of the plasma membrane to aless hydrophobic membrane domain.
 4. The method of claim 1 where themodification is a redistribution of G_(sα) from cell processes andprocess tips to the cell body.
 5. The method of claim 1 wherein theperipheral tissues are blood cells
 6. The method of claim 5 wherein theblood cells are erythrocytes.
 7. The method of claim 5 wherein the bloodcells are leukocytes.
 8. The method of claim 5 wherein the blood cellsare platelets.
 9. The method of claim 1 wherein the peripheral tissuesare skin fibroblasts.
 10. A method for detecting the effectiveness ofantidepressant therapy in a depressed individual, the method comprising(a) collecting cells from peripheral tissues from the depressedindividual; and (b) determining whether there has been a modification ofthe association of G_(sα) with components of the plasma membrane orcytoskeleton of the cells collected in step (a).
 11. The method of claim10 wherein the modification is enhanced coupling between G_(sα) andadenylyl cyclase.
 12. The method of claim 10 wherein the modification isa redistribution of G_(sα) from a strongly hydrophobic region of theplasma membrane to a less hydrophobic membrane domain.
 13. The method ofclaim 10 where the modification is a redistribution of G_(sα) from cellprocesses and process tips to the cell body.
 14. The method of claim 10wherein the peripheral tissues are blood cells.
 15. The method of claim14 wherein the blood cells are erythrocytes.
 16. The method of claim 14wherein the blood cells are leukocytes.
 17. The method of claim 14wherein the blood cells are platelets.
 18. The method of claim 10wherein the peripheral tissues are skin fibroblasts.
 19. A method forassaying for an agent or agents having antidepressant activitycomprising the step of: (a) contacting said agent or agents withcultured cells expressing Type VI adenylyl cyclase; (b) determiningwhether there has been a modification of the association of G_(sα) withcomponents of the plasma membrane or cytoskeleton of the cells in step(a) via comparison to a control cell culture lacking said agent oragents; (c) identifying agents having antidepressant activity from adifference in the modification of the association of G_(sα) withcomponents of the plasma membrane or cytoskeleton of the cells in step(a), wherein an agent or agents having antidepressant activity increasesthe modification of the association of G_(sα) with components of theplasma membrane or cytoskeleton of the cells in step (a).
 20. The methodof claim 19 wherein the modification is enhanced coupling between G_(sα)and adenylyl cyclase.
 21. The method of claim 19 wherein themodification is a redistribution of G_(sα) from a strongly hydrophobicregion of the plasma membrane to a less hydrophobic membrane domain. 22.The method of claim 19 where the modification is a redistribution ofG_(sα) from cell processes and process tips to the cell body.
 23. Themethod of claim 19 wherein the peripheral tissues are blood cells. 24.The method of claim 23 wherein the blood cells are erythrocytes.
 25. Themethod of claim 23 wherein the blood cells are leukocytes.
 26. Themethod of claim 23 wherein the blood cells are platelets.
 27. The methodof claim 19 wherein the peripheral tissues are skin fibroblasts.
 28. Themethod of claim 19 wherein the cultured cells are of neuronal or glialorigin.
 29. The method of claim 19 wherein the cultured cells arecultured epithelial cells expressing Type VI adenylyl cyclase.
 30. Amethod for assaying for an agent or agents having the ability to modifythe association of G_(sα) with components of the plasma membrane orcytoskeleton of cells comprising the step of: (a) contacting said agentor agents with cultured cells expressing Type VI adenylyl cyclase; (b)determining whether there has been a modification of the association ofG_(sα) with components of the plasma membrane or cytoskeleton of thecells in step (a) via comparison to a control cell culture lacking saidagent or agents; (c) identifying agents having antidepressant activityfrom a difference in the modification of the association of G_(sα) withcomponents of the plasma membrane or cytoskeleton of the cells in step(a), wherein an agent or agents having antidepressant activity increasesthe modification of the association of G_(sα) with components of theplasma membrane or cytoskeleton of the cells in step (a).
 31. The methodof claim 30 wherein the modification is enhanced coupling between G_(sα)and adenylyl cyclase.
 32. The method of claim 30 wherein themodification is a redistribution of G_(sα) from a strongly hydrophobicregion of the plasma membrane to a less hydrophobic membrane domain. 33.The method of claim 30 where the modification is a redistribution ofG_(sα) from cell processes and process tips to the cell body.
 34. Themethod of claim 30 wherein the peripheral tissues are blood cells. 35.The method of claim 34 wherein the blood cells are erythrocytes.
 36. Themethod of claim 34 wherein the blood cells are leukocytes.
 37. Themethod of claim 34 wherein the blood cells are platelets.
 38. The methodof claim 30 wherein the peripheral tissues are skin fibroblasts.
 39. Themethod of claim 30 wherein the cultured cells are of neuronal or glialorigin.
 40. The method of claim 30 wherein the cultured cells arecultured epithelial cells expressing Type VI adenylyl cyclase.